The Molecular Core of the YCCMD provides support for a number of specialized methodologies and technical assistance in essential molecular techniques. The creation of animal models of gene dysregulation and the characterization of target gene expression is fully supported the Molecular Core, with further analysis both in vivo and in vitro provided by the Cell and Physiology Cores. Aim 1. To provide as services, a) the construction of transgenes and gene disruption vectors, and gene targeting vectors and conditional gene targeting vectors using the cre-/loxP method. Genotyping is carried out by PCR or Southern blot analysis of tail DNA. Aim 2. To provide as services, a) the localization of gene expression by in situ hybridization, and b) the quantitation of gene expression by RNase protection. This includes the dissection and fixation of tissues, embedding in paraffin or OCT, sectioning by microtome or cryostat, preparation of riboprobes or oligoprobes, hybridization, emulsion autoradiography and image analysis. Aim 3. To provide technical assistance for both basic and advanced methods in molecular biology, which range from hands-on training to consultative assistance with experimental design, data interpretation and troubleshooting. Supported methods include, Southern and Northern blotting; RNA and genomic DNA preparation and analysis; stable or transient cell transfection by calcium phosphate, liposome or electroporation; reporter gene analysis using CAT, luciferase or GFP; the construction of vectors for the expression of proteins in bacteria or in eukaryotic cell lines using dominant selectable markers; cDNA cloning using oligonucleotides deduced from peptide sequence, expression cloning, cDNA and genomic library construction; and competitive PCR.